The MHC is the most polymorphic locus in the human genome, and it is also the richest segment genome wide in terms of disease associations, including bacterial, viral, inflammatory, and autoimmune diseases, cancer, neurologic disorders, and drug hypersensitivity. Assigning the mechanism for these disease associations is confounded by the extensive polymorphism, dense gene content with strong linkage disequilibrium between pairs of genes, and functional similarity of the molecules encoded by genes within the MHC. The regulation of HLA class I expression is mediated by a series of core promoter motifs, which are located within 500 bp upstream of the start site and are relatively conserved. Binding sites for regulatory proteins further upstream from the core promoter have also been identified and it is likely that others exist, some of which may be polymorphic. Indeed, genetic variants and epigenetic modifications located within the 3 and 5 untranslated regions (UTRs), as well as the coding region, have been shown to alter HLA allele expression levels, but so far, these mechanisms account for only a small percentage of the expression variation found within the respective genes. Differential HLA expression levels have recently been shown to associate with a number of diseases. We determined sequences extending nearly 2 kb upstream of the transcription start site for 68 alleles from 57 major lineages of classical HLA class I genes. The nucleotide diversity within this promoter segment roughly follows that seen within the coding regions, with HLA B showing the highest (approx.1.9%), followed by HLA A (approx.1.8%), and HLA C showing the lowest diversity (approx. 0.9%). Despite its greater diversity, HLA B mRNA expression levels determined in 178 European Americans do not vary in an allele or lineage specific manner, unlike the differential expression levels of HLA A or HLA C reported previously. Close proximity of promoter sequences in phylogenetic trees is roughly reflected by similarity of expression pattern for most HLA A and C loci. Although promoter sequence divergence might impact promoter activity, we observed no clear link between the phylogenetic structures as represented by pairwise nucleotide differences in the promoter regions with estimated differences in mRNA expression levels for the classical class I loci. Further, no pair of class I loci showed coordinated expression levels, suggesting that distinct mechanisms across loci determine their expression level under nonstimulated conditions. These data serve as a foundation for more in depth analysis of the functional consequences of promoter region variation within the classical HLA class I loci. HLA class I peptide loading involves several proteins including tapasin, whose function is editing the peptide repertoire presented by HLA class I. Experimental data demonstrate differential dependence of HLA class I allotypes on tapasin to form stable peptide complex. In tapasin deficient cell lines, some HLA class I allotypes can be expressed at normal levels on the cell surface, whereas others are almost undetectable. Remarkably, two alleles, representing protective and susceptible alleles in HIV disease demonstrate opposite phenotypes: tapasin dependent B*57:01 and tapasin independent B*35:01, respectively. Given the importance of tapasin in assembly of certain HLA allotypes and its potential influence on peptide repertoire, we hypothesized that genetically defined variation in tapasin/HLA interactions may influence HIV disease outcomes. Recently, a set of 27 HLA B alleles were tested in Dr. Malini Raghavan's lab at the University of Michigan, in the tapasin-deficient M553 cell line, which demonstrated a wide range of expression levels. These expression levels can be used as characteristics of tapasin dependence of the HLA class I alleles and serve as variables in statistical analysis. To optimize the analysis, we expanded the Raghavan dataset to 42 HLA B alleles,which represent all HLA B alleles with frequencies more than 0.5 percent in either white or black populations. The alleles were expressed in the 721.220 tapasin negative B cell line using lentiviral constructs and the expression levels were measured by FACS. Each HLA class I allele was assigned tapasin dependence value based on these expression levels (the higher expression the more independent the corresponding allele is). Variation in the gene encoding tapasin, includes one non-synonymous variation (rs2071888) corresponding to two versions of protein with arginine and threonine at position 240 of a mature peptide. The variants are almost equally presented among whites, whereas Arg is more common in blacks (72% allelic frequency). Molecular dynamic simulations demonstrate that the variation at pos 240 may influence interactions with ERp57, another component of the peptide loading complex, and HLA class I. We have genotyped rs2071888 in our HIV cohorts and our preliminary analyses indicate that Thr240 associates with lower viral load among individuals with tapasin dependent alleles. We are planning to expand the genotyping dataset to increase the statistical power. In addition, we will characterize further tapasin dependence by reconstituting expression of both versions of tapasin (Thr/Arg240) in 721.220 cells. We will also perform similar experiments in M553 cell line to see how different cells can influence tapasin dependence.